Ligand Sulfur Oxidation State Progressively Alters Galectin-3-Ligand Complex Conformations To Induce Affinity-Influencing Hydrogen Bonds

Galectins play biological roles in immune regulation and tumor progression. Ligands with high affinity for the shallow, hydrophilic galectin-3 ligand binding site rely primarily on a galactose core with appended aryltriazole moieties, making hydrophobic interactions and π-stacking. We designed and synthesized phenyl sulfone, sulfoxide, and sulfide-triazolyl thiogalactoside derivatives to create affinity-enhancing hydrogen bonds, hydrophobic and π-interactions. Crystal structures and thermodynamic analyses revealed that the sulfoxide and sulfone ligands form hydrogen bonds while retaining π-interactions, resulting in improved affinities and unique binding poses. The sulfoxide, bearing one hydrogen bond acceptor, leads to an affinity decrease compared to the sulfide, whereas the corresponding sulfone forms three hydrogen bonds, two directly with Asn and Arg side chains and one water-mediated to an Asp side chain, respectively, which alters the complex structure and increases affinity. These findings highlight that the sulfur oxidation state influences both the interaction thermodynamics and structure.


■ INTRODUCTION
Structural and thermodynamic analyses are of key importance for understanding target protein−ligand interactions and, thus, for facilitating drug discovery.Identifying and characterizing potential sites for H-bonds, π interactions, ionic, and other polar interactions between protein and ligand 1−3 is at the center of this process.A common strategy to improve affinity between a ligand and a protein is to introduce hydrophobic, typically aromatic, structural moieties in a lead structure to interact with, e.g., hydrophobic, aromatic, and/or cationic parts of the protein site.However, this approach may lead to overly hydrophobic molecules with potentially less favorable absorption, distribution, metabolism, and excretion (ADME) properties and poor solubility.Therefore, introducing polar interactions during lead optimization can be advantageous to enhance the affinity.
An ideal model for studies of ligand−protein interactions is galectin-3, which belongs to a family of lectins having a natural affinity for β-D-galactopyranosides. 4 The choice of galectin-3 is advantageous for studies of protein−ligand interactions, as it is easily expressed, stable in solution to high concentrations, amenable to nuclear magnetic resonance (NMR) spectroscopic experiments, and crystallizes and diffracts to high resolution with ligands complexed.−8 For example, the flexible side chain Arg144 has been demonstrated to play an important role in ligand binding. 9,10In the presence of inhibitors equipped with aryl moieties at C3 of the galactose moiety, Arg144 rearranges from its location on the surface of the galectin-3 carbohydrate recognition domain (CRD), in a water-mediated salt bridge to Asp148, to a new position in which it creates a binding pocket partly mediated by cation−π interactions with the ligand aryl group. 11More recently, we have carried out several systematic studies of the interactions of arene moieties in synthetic ligands with Arg144, in particular how the choice of different aromatic groups or substituents influences the affinity. 3,6,9,12Recent studies of solvation and fluorination effects on the Arg144arene interactions with a series of phenyltriazole-derivatized galactosides 6 raised the question of whether the conformation of Arg144, and consequently the ligand affinity, could be further influenced by introducing hydrogen bonding functionalities in the ligand structure.The arene−Arg144 interaction has been exploited in the development of topical 13,14 and orally 8,15 administered drugs currently in clinical trials.Herein, we present the synthesis of a sulfide-sulfoxide-sulfone series of galectin-3 ligands together with evaluations of their affinity and binding thermodynamics combined with high-resolution structural analysis in order to understand hydrogen-bondassisted aryl−arginine face-to-face stacking in galectin-3-ligand complexes.The results suggest that arene−Arg144 and hydrogen bond interactions can be tuned by changing the ligand sulfur oxidation state, leading to affinity enhancement and galectin-3 ligand site reorganization to novel ligand-bound conformations.

■ RESULTS AND DISCUSSION
Synthesis and Affinity Evaluations.Initially, a series of analogous galactosides derivatized at the pyranose C3 position with phenylsulfide 4a, sulfoxide 4b, and sulfone 4c at the triazole C4 position was synthesized (Scheme 1) and evaluated and compared to the phenyltriazole derivative 1 for galectin-3 interaction characteristics (Table 1).Synthesis of compounds 4a−4c was done from the known p-methylphenyl 3-azido-3deoxy-β-D-thiogalactopyranoside 2 6 via CuI-mediated 1,3dipolar cycloaddition with (trimethylsilylethynylsulfanyl)benzene 3a, trimethylsilylethynyl phenyl sulfoxide 3b, and trimethylsilylethynyl phenyl sulfone 3c, respectively, and with spontaneous in situ desilylation (Scheme 1).Evaluation of the galectin-3 affinities of these ligands in a competitive fluorescence polarization assay (Table 1) showed that the sulfide 4a bound more weakly to galectin-3 than the reference, 1 with a K d of 260 μM compared to 88 μM for the reference.The racemic sulfoxide 4b had an even weaker affinity (K d 600 μM), while the sulfone regained and marginally improved affinity over that of 1, with K d of 79 μM.The phenyltriazole moiety has been shown to interact with Arg144 in galectin-3; thus, to gain an understanding of why the phenylsulfone 4c regained affinity, it was evaluated for affinity against the galectin-3 R144S 13,16 and R144K 13 mutants in the fluorescence polarization assay.A critical role for the Arg144 side chain was revealed, as 4c bound more weakly to both mutants, with K d values of 600 μM for R144S and 250 μM for R144K.The somewhat higher affinity of R144K than that of R144S may indicate that a cationic and/or larger side chain forms beneficial interactions with the phenyl sulfone moiety of 4c.
Initial attempts at crystallographic structural analyses of galectin-3C (the C-terminal domain of galectin-3) with 4a, 4b, and 4c failed due to insufficient solubility of all compounds.Hence, we adopted a strategy to increase the ligand solubility by replacing the hydrophobic tolyl aglycon with a glucose residue.The known thiodiglycoside 5 6 was treated with 3a and 3b and 3c in the presence of DIPEA and CuI to afford the corresponding disaccharides 6a, 6b, and 6c, respectively (Scheme 2).Affinity evaluations revealed higher affinities of 6a−6c for galectin-3 but with the same affinity trends among the three ligands and toward the reference phenyltriazole 7 3 (Table 2).Scheme 2. Synthesis of the Thiogalactoglucosides 6a, 6b, and 6c and the Structure of the Reference Phenyltriazole 7

Journal of Medicinal Chemistry
Structural Analysis of Galectin-3C in Complex with 6a, 6b, and 6c.High-resolution X-ray structures of 6a, 6b, and 6c in complex with galectin-3C were obtained at 1.08− 1.14 Å resolution (Table 3).In all three complexes, the protein atoms superimpose very well, and there are no significant conformational differences in the protein in the vicinity of the binding site apart from Arg144 (Figure 1).The binding modes of the galactose and glucose moieties are identical to previously reported structures 3,6 (Figure 1).However, the C3 substituents provide two novel binding modes with Arg144 that depend on the oxidation state of the sulfur atom, neither of which has previously been reported for galectin-3 inhibitors.One face of the phenyl groups of the sulfoxide 6b and sulfone 6c is directed toward the solution, resulting in some flexibility, which is reflected by slightly poorer electron density for this moiety compared to the rest of the ligand, especially for the phenyl group of the sulfoxide 6b (Figure 1C).
In the 6a complex, the introduction of a sulfur atom as a spacer between the triazole and phenyl moieties, compared to the known compound 7 3 where the phenyl and triazole rings were directly linked with a bond, results in an inward shift of the entire C3-substituent toward the protein by about 1 Å (Figure 2A).In 6a, the sulfur atom occupies approximately the same position as the ortho-carbon atom in the unsubstituted phenyl group of the published phenyltriazole 7. The dihedral angles of the C−S−C bond orient the phenyl group in a new position that causes an upward displacement of the side chain of Arg144 to an orientation different from that of Arg144 in complex with 7, where Arg144 sandwiches the phenyltriazole moiety between its side chain and that of Ala146.In the 6a complex, Arg144 is further moved upward to maintain a cation−π interaction with the phenyl group of 6a, albeit with a poorer overlap between the phenyl group and the guanidinium moiety of Arg144, as the interaction is shifted toward the aliphatic part of the Arg144 side chain (Figure 2A).
The oxidation of the sulfur atom in 6a to the sulfoxide 6b renders the sulfur atom a chiral center and synthesis produces a mixture of R-and S-enantiomers (Figure 2B,D).The electron density for 6b in the complex is compatible with a mixture of both enantiomers, both of which have an oxygen atom oriented toward the protein.It is difficult to establish the exact ratio of the two enantiomers and they have been modeled with 50% occupancy each.Surprisingly, 6b also induces a new conformation of Arg144 that has not been seen in any galectin-3C structure to date.In this conformation, Arg144 is sandwiched between the phenyl group of 6b and the π-system of the amide bond of Ser237-Gly238 (Figure 3).In the previous phenyltriazole ligand series, Ser237-Gly238 represented the inner boundary of the aryl binding pocket that was exploited for fluorine-amide interactions with the main chain atoms of these two residues. 3In this novel position, the guanidinium group of Arg144 makes two hydrogen bonds: one to the side chain hydroxyl of Ser237 and the other to the sulfoxide oxygen of S-6b.The binding mode of the sulfone 6c is very similar to that of the sulfoxide 6b (Figure 2C,D), which is logical since the sulfur atom has tetrahedral geometry in both cases, and 6c thus mimics the simultaneous binding of both enantiomers of 6b.The sulfoxide and sulfonyl groups of S-6b and 6c, respectively, together with the triazole of all compounds, coordinate a water molecule that bridges to the Asp148 side chain, but the distances between the water molecules, residues, and the ligand vary (Figure 2C,D).In the case of sulfide 6a, only the triazole nitrogen binds the water molecule (Figure 1D) at 3.3 Å from the triazole nitrogen and 2.7 Å from Asp148.For sulfoxide 6b, the water molecule is 2.9 Å from the sulfoxide oxygen of S-6b and 2.7 Å from Asp148 (Figures 1E and 2B,C).In 6c, the water molecule is 2.6 Å from the nearest sulfone oxygen and 2.7 Å from Asp148 (Figure 1F).The introduction of a second oxygen on the sulfur atom in 6c also pushes the water molecule toward bulk solvent, thus increasing the distance between Asp148 and the water molecule.Furthermore, the sulfoxide oxygen in S-6b forms an H-bond with Arg144 and the water molecule, and R-6b sulfoxide oxygen is placed near N160, albeit at a less-thanoptimal interaction distance and angle.
Thermodynamic Analysis.Isothermal titration calorimetry (ITC) of 6a−6c with galectin-3C was performed to gain a

Journal of Medicinal Chemistry
further understanding of the thermodynamics of binding of 6a−6c to galectin-3.Galectin-3C was titrated into the ITC cells containing 6a−6c (Table 4 and Figure 4).The enthalpies of binding of the sulfide 6a and sulfoxide 6b were −6.17 ± 0.15 and −6.15 ± 0.39 kcal/mol, respectively, whereas the TΔS terms were 0.29 and 0.62 kcal/mol, which translates to K d values of 55.5 ± 7.89 and 94.2 ± 3.7 μM for 6a and 6b, respectively.The higher affinity of 6c was confirmed by an enthalpic contribution of −10.5 ± 1.5 kcal/mol and TΔS of 3.4 kcal/mol, giving K d of 6.2 ± 0.18 μM.Hence, the ITC affinities of 6a−6c correlate overall with those obtained with the competitive fluorescence polarization assay.The higher affinity of 6c is primarily a result of enhanced enthalpy of binding compared to 6a−b, albeit with an accompanying larger entropy penalty, which may be a consequence of added hydrogen bonds to galectin-3C.The sulfoxide 6b also forms hydrogen bonds to galectin-3C, but the two diastereomers do so with different residues; R-6b binds the Asn160 side chain amide, while S-6b binds directly to Arg144 and to Asp148 via a water molecule (Figure 3).One may speculate that the two different hydrogen bonding patterns of the two diastereomers of 6b display different thermodynamic characteristics that may at least partly counteract each other in the ITC analysis of the 6b mixture.The sulfone 6c forms all hydrogen bonds to Asn160, Arg144, and water-mediated to Asp148, which is likely reflected in the significantly greater enthalpy of binding compared to 6a and 6b but also in the greater entropic penalty.

■ CONCLUSIONS
Sulfide, sulfoxide, and sulfone-derivatized galectin-3-binding ligands 6a−c were synthesized, and their binding thermody-namics and complex structures were analyzed.According to Xray structural analysis, a sulfoxide diastereomeric mixture of the disaccharide ligand 6b formed hydrogen bonds to galectin-3C not seen for the parent sulfide 6a, but this resulted in no significant influence on the thermodynamics of binding, and the sulfoxide displayed a somewhat weaker affinity.A possible explanation may be that one of the 6b diastereomers binds significantly weaker than the other, resulting in a weak affinity of the 6b diastereomeric mixture.On the other hand, sulfone 6c formed two additional hydrogen bonds to galectin-3 compared to sulfide 6a, which translated to a significantly more favorable enthalpy of binding and higher affinity.Simultaneously to the stepwise addition of ligand-galectin-3C hydrogen bonds in the sulfide-sulfoxide-sulfone series 6a−6c, a sulfur-linked ligand phenyl shifted its position in the complex with galectin-3 which led to an accompanying gradual movement of the Arg144 side chain position.These results emphasize the significance of the sulfur oxidation state in optimizing the interaction and structure of galectin-3 ligands, opening new avenues for developing pharmaceuticals targeting galectins.
To a suspension of 2 (20 mg, 0.046 mmol), CuI (1 mg, 10 mol%), and 3b (10 mg, 0.069 mmol) in DCM (3 mL), DIPEA (15 μL, 0.069 mmol) was added.The mixture was stirred at room temperature for 16 h, filtered, and washed with methanol (10 mL).The solvent was concentrated and the residue was purified with flash chromatography (SiO 2 , 5% MeOH in DCM), followed by preparative HPLC to afford 4b (17 mg, 61%) as white amorphous solid.FLUOstar Galaxy software or with a PheraStarFS plate reader and PHERAstar Mars version 2.10 R3 software (BMG, Offenburg, Germany).The dissociation constant (K d ) values were determined in PBS as described earlier. 13,18Compounds were dissolved in neat DMSO at 20 mM and diluted in PBS to 3−6 different concentrations to be tested in duplicate.Average K d values and SEMs were calculated from 2 to 8 single-triple point measurements showing between 20 and 80% inhibition.
Co-Crystallization of Galectin-3C with Compounds 6a, 6b, and 6c.Small crystals of lactose-bound galectin-3C were grown with the hanging drop method in NeXtal plates (NeXtal Biotechnologies) with the following reservoir condition: 20% (w/v) PEG 4000, Tris-HCl pH 7.5, 0.4 M NaSCN, 10 mM β-mercaptoethanol, as described previously. 5Small crystals were then moved to drops containing the same reservoir with the addition of 10 mM of the ligand (6a, 6b, and 6c), from a 100 mM stock solution in DMSO.Soaking lasted for 12− 15 h.10% PEG 400 was added as a cryoprotectant just before freezing the crystals.Soaked crystals were then frozen in liquid nitrogen.
Data Collection and Structure Solution of Galectin-3C in Complex with 6a, 6b, and 6c.Data were collected at the BioMAX beamline of the MAX IV synchrotron in Lund, Sweden.For each complex, 3600 diffraction images were collected with a 0.1°rotation and 11 ms exposure time.All data were integrated using XDS.Scaling and merging were done with Aimless. 19Cross-validation of refinement was based on 10% of the reflections.Molecular replacement was done using Phaser in the Phenix suite 20 version 1.14 using the highresolution lactose − galectin-3C structure 5 (PDB ID 3ZSJ), with lactose and water molecules removed, as the template.Ligands and their crystallographic restraints were generated through phenix.eL-BOW. 21Restrained refinement was then performed using phenix.refine.Manual rebuilding, including the addition of water molecules, was done using Coot. 22All of the images were made with PyMOL (Schrodinger LLC).
Isothermal Titration Calorimetry.All ITC experiments were performed on a MicroCal PEAQ-ITC (Malvern) at 301 K with 11 injections of 2.5 μL per injection (first injection, 0.4 μL) of the ligand into the protein.Stock solutions of the ligands were prepared in a buffer at 5 mM.The ligands were diluted with buffer to concentrations between 2 and 3 mM.All experiments were performed with galectin-3C concentrations between 0.19 to 0.25 mM in the cell.All thermograms were integrated using NITPIC, the titration curves were fitted using SEDPHAT with error estimates using the automatic confidence interval search with projection method, and all figures were made in GUSSI.
The crystal structures of galectin-3C in complex with compounds 6a, 6b, and 6c have been deposited in the Protein Data Bank (http://wwpdb.org)with accession numbers 8PBF, 8PF9, and 8PFF.The structures will be released on article publication (PDF) Molecular formula strings (SMILE) as a csv file (CSV)

Figure 1 .
Figure 1.(A) Surface view of the galectin-3C complexes with compounds 6a, 6b, and 6c.Arg144 is labeled.Compounds are shown in stick representation.Compound 6a (PDB ID 8PBF) is shown with carbon atoms colored green, 6b (8PF9) cyan, and 6c (8PFF) magenta, respectively.The same color scheme is used in all subsequent panels.(B) Details of the binding mode for the compounds showing all of the key residues in galectin-3.The carbon atoms of the side chain of Arg144 are colored according to the respective ligand to show its two conformational states.The conformation for 6b is not visible as it is identical to and thus masked by that of 6c.Other side chains do not vary in conformation between the complexes.(C) 2 | F o | − | F c | electron density for the compounds.The R-and S-enantiomers of 6b are shown separately in the same map.The maps are contoured at a level of 1σ above the mean.(D−F) Individual binding modes and polar contacts of 6a−6c.The ligands are colored as in (C).Panel E shows both enantiomers of 6b, which are identical except at the sulfur atom; therefore, the interactions of both enantiomers are shown.The differences between these are visualized in Figure 3.

Figure 2 .
Figure 2. Closeup view of the binding of the compounds near Arg144 in the binding pocket.(A) Comparison of the sulfide 6a (PDB ID 8PBF, green) and unsubstituted phenyltriazole 7 (pink).(B) Sulfide 6a (green) and the sulfoxide 6b (8PF9, both R and S enantiomers shown) (cyan).(C) Sulfide 6a (green) and the sulfone 6c (8PFF.magenta).(D) Sulfoxide 6b (R and S) (cyan) and the sulfone 6c (magenta).Key polar contacts are shown and color-coded green for 6a, cyan for 6b (R and S), and magenta for 6c.Key water molecules in each structure are shown as small spheres colored the same way as the carbon atoms of the ligand.However, all water molecules are omitted from panel A for clarity.Hydrogen bonds made by the ligands are shown as dashed lines colored as for the C atoms.Hydrogen bond distances are shown in Å.

Figure 3 .
Figure 3. Binding modes of the S and R configurations of 6b (PDB ID 8PF9).The S-configuration sulfoxide oxygen coordinates Arg144 and a water molecule together with Asp148.The R-configuration sulfoxide oxygen makes a long 3.4 Å contact at a nonideal angle with Asn160.Key distances and polar contacts are shown.

Table 3 .
Data Collection Statistics and Model Quality for Crystal Structures of Galectin-3C in Complex with 6a−6c a a Numbers in parentheses describe the highest-resolution shell.